Antibody Drug Conjugates (ADC) are compounds for the targeted delivery of payloads to a target. In many instances, the target is a tumor associated antigen (TAA) and the payload is a drug.
There are a variety of classes of antibodies including IgG1 and IgG2. The structure of an IgG1 and IgG2 antibody includes 2 heavy chains (HC) and 2 light chains (LC). The two heavy chains and two light chains presented as a complex comprise an intact antibody. Each IgG1 antibody has 12 intrachain and 4 interchain disulfide bonds created through the oxidation of their respective cysteines on each antibody chain. There are two disulfide bonds between the heavy chain and light chains and two disulfide bonds between the heavy chain and the heavy chain. Complete reduction of the interchain disulfide bonds of an IgG1 antibody results in the antibody complex being maintained through non covalent interactions.
Complete reduction of the interchain disulfide bonds of an IgG1 antibody yields eight accessible thiols for conjugation with a drug, generally via a linker. By varying the reduction parameters, antibody isomers with from 0 to 8 thiols available for conjugation can be obtained. For example, reducing an IgG1 with DTT can yield antibodies with 2, 4, 6, or 8 accessible thiols. Fully reduced ADCs are held together by non-covalent interactions, such as hydrogen bonds, ionic bonds, hydrophobic, and van der Waal's interactions, and will separate into light and heavy chains under denaturing conditions (e.g., reverse phase chromatography). Non fully reduced ADCs are held together by covalent and non covalent interactions. The portions of non-fully reduced ADCs held together by covalent means may also be separated under denaturing conditions.
Fully loaded IgG1 ADCs have one drug molecule attached to each cysteine that makes up the interchain disulfides of an antibody for a total of eight drugs per antibody. Partially loaded ADCs generally have 2, 4, or 6 drug molecules attached to cysteine residues. Partially loaded ADCs are also observed to have 1, 3, and 5 drug molecules attached to cysteine residues. While the mass of the constituent fragments of loaded ADCs have been assayed by mass spectrometry (MS), there remains a problem in the field to which the present invention relates regarding assaying the mass of the intact loaded ADC.
Although techniques for directly measuring the mass of an intact loaded ADC are lacking, various indirect means for measuring the mass of loaded ADCs are known. For example, the mass of an ADC has been measured by binding a sample (e.g., an ADC) to a heated reversed phase-high pressure liquid chromatographic column (rp-HPLC) in the presence of low or no organic solvent which typically also contains an ion pairing acid (e.g., trifluoracetic acid) and allows the non-volatile salts and surfactants to be washed from the sample (commonly referred to as desalting). In this example, the protein is eluted from the rp-HPLC column by increasing the organic content of the solvent to the point at which the interactions between the hydrophobic protein domains and the surface of the rp-HPLC column are disrupted by a non-polar organic solvent. An undesirable effect of this technique is that it destroys the protein structure by subjecting the protein samples to heat, acid and organic solvent, any one of which can denature proteins and destroy the protein structure. When non-covalent protein complexes are subjected to rp-HPLC they fall apart into their constituent covalent entities. the case of an IgG1 antibody with 8 interchain linked cysteine drugs, desalting on a rp-HPLC column results in complete dissociation of the ADC into heavy chains with 3 drugs per chain and light chains with 1 drug per chain. While the mass of the constituent fragments can be determined by mass spectrometry (MS), techniques for determining the mass of the intact entity are lacking in the field to which the present invention pertains.
Current MS techniques are lacking for measuring the mass of intact ACDs due, in part, to the fact that these techniques lead to protein denaturation and/or are too time consuming for the uses contemplated herein. Virtually all methods of native electrospray ionization (ESI) MS of proteins specify that this procedure should be carried out at nanospray scale (flow rate of 100 to 500 nanoliters/minute) to minimize the disruption to protein structure that would occur from heated sheath and desolvation gases that are used for standard ESI. The sample handling process for measuring the native mass of an ADC using conventional nanospray ESI-MS techniques is very time consuming and not amenable to high throughput. Not including the time to deglycosylate the antibody, it takes at least an hour per sample to obtain a mass measurement,
Furthermore, previously known methods for analyzing interchain cysteinyl-linked ADC's are either not amenable to on-line mass spectrometry HIC) or result in the denaturing dissociation of conjugated heavy and light chains during chromatographic separation and subsequent mass measurement (e.g., rp-HPLC). Therefore, there is a need in the field to which the instant invention pertains related to methods for routinely and rapidly determining the intact mass of a cysteine-linked ADC.
Surprisingly, the present invention meets this need as well as other unmet needs in the relevant field by providing methods, devices, and systems for detecting the mass of a non covalently associated protein agent conjugate.